Biodegradable polymeric nanocapsules and uses thereof

ABSTRACT

The present invention relates to a biodegradable polymeric nanocapsule composition, adaptable for encapsulation of an agent of therapeutic interest for enhancing the in vivo circulation time of thereof and uses thereof.

BACKGROUND OF THE INVENTION

(a) Field of the Invention

The invention relates to the field of nanocapsule compositions havingimproved circulation times, uses thereof, and methods for preparing thesame. The nanocapsule compositions of the present invention areadaptable to encapsulate agents of therapeutic interest, includingmacromolecules.

(b) Description of Prior Art

Hemoglobin (Hb) in red blood cells (RBC) is responsible for transportingoxygen. When extracted from a RBC, Hb can be sterilized to remove H.I.V.and other infective agents. Unfortunately, when the extracted Hb isinfused into the body, it breaks down into dimers after infusion intothe circulation of a recipient. Free Hb is also toxic especially for thekidney. Hemoglobin molecules can be chemically modified to preventdegradation after infusion. These simple modified hemoglobins are in thefinal stages of human testing. However, this type of modified hemoglobinis not covered by a membrane and as a result, it has to be ultra-pure toavoid adverse reactions. This also removes all the red blood cellenzymes that are needed to prevent the damaging effects of oxidants.Furthermore, the circulation half-time of modified hemoglobin in humanis rather short, at approximately 24 hours.

Hemoglobin is but one example of the many biologically importantmacromolecules required by the body. In the event of illness, it isoften desirable to supplement the body with macromolecules that areeither lacking as a result of the illness or identified to have atherapeutic effect. Unfortunately, however, it has been difficult in thepast to adequately deliver such macromolecules into the body in anacceptable manner to obtain the desired therapeutic effect.

In the past, attempts to microencapsulate hemoglobin for in vivo usehave been made (Chang, T. M. S., 1964, Science 146, 524; Chang, T. M.S., 1997, “Blood Substitutes:Principles, methods, products and clinicaltrial”, vol. I, Karser publisher, Basel). Collodion, cellulose, HMDA(1,6-hexamethylenediamine), cross-linked protein, bilayer ofphospholipid-cholesterol complexed on cross-linked protein membrane andother materials have been used to coat droplets of hemoglobin solution(Chang, T. M. S., 1997, “Blood Substitutes:Principles, methods, productsand clinical trial”, vol. I, Karser publisher, Basel). However, theseartificial cells with diameters of about one micron survived for a veryshort time in the host circulation following intravenous injections.Furthermore, the polymer membrane of these artificial cells accumulatedin the body.

Emphasis then turned to the use of phospholipids in the preparation ofliposomes containing hemoglobin (Chang, T. M. S., 1997, “BloodSubstitutes:Principles, methods, products and clinical trial”, vol. I,Karser publisher, Basel). The use of submicron phospholipid-cholesterolmicrocapsules (liposomes) increased the survival time of hemoglobin inthe circulation (Djordjevich, L. et al., 1980, Exp. Hematol. 8, 584).The drawback to these liposomes is their insufficient stability andstrength and also the sensitivity of the phospholipid membranes toenvironmental degradation. Liposomes are subject to degradation duringstorage and while in host circulation. Furthermore, the lipid membranesare removed and accumulated in cells that are normally needed to removebacteria and toxin from the circulation. As a result, the body's abilityto fight infection and toxin can be markedly reduced.

Subsequently, a biodegradable polymer membrane containing hemoglobin wasdeveloped, as described in Applicant's U.S. Pat. No. 5,670,173, which isherein incorporated by reference. Here, a biodegradable polylactidemembrane containing hemoglobin of about 150 nanometre diameter wasprepared (T. M. S. Chang and W. P. Yu, U.S. Pat. No. 5,670,173 issued onSep. 23, 1997). Polylactide can be readily converted to water and carbondioxide after use and therefore does not accumulate in the body.

On average, after infusion, the polymer membrane as disclosed in U.S.Pat. No. 5,670,173 circulates with a half-time of less than 2 hours. Ithas been subsequently determined that for practical purposes, such asblood substitutes, for example, a biodegradable polymer membrane havinga longer circulation half-time is preferred.

Accordingly, it would be highly desirable to be provided with amulti-purpose biodegradable nanocapsule having an improved in vivocirculation time.

It would also be highly desirable to be provided with a biodegradablenanocapsule having an effective circulation time of at least 6 hours.

It would be further desirable to be provided with a biodegradablenanocapsule having an effective circulation time of at least 14 hours.

It would be further desirable to be provided with a biodegradablenanocapsule having an effective circulation time of at least 24 hours.

It would be yet further desirable to be provided with a multi-purposebiodegradable nanocapsule that is selectively permeable to therapeuticagents of interest.

It would also be desirable to be provided with a multi-purposebiodegradable nanocapsule that is adaptable for the encapsulation of anagent of therapeutic interest, and delivery thereof in vivo.

It would be further desired to be provided with a nanocapsulecomposition adaptable to deliver an encapsulated agent of therapeuticinterest in vivo with a controlled rate of release.

SUMMARY OF THE INVENTION

The present invention provides a multi-purpose biodegradable nanocapsulehaving an improved in vivo circulation time. The biodegradablenanocapsules of the present invention are adaptable for encapsulating anagent of therapeutic interest and subsequently delivering the same invivo. The biodegradable nanocapsules of the present invention may beemployed to encapsulate a plurality of agents of therapeutic interest,including, without limitation, macromolecules, such as hemoglobin,enzymes, polypeptides, genes, and polymerized proteins and enzymes suchas polyhemoglobin etc.

Preferably, the biodegradable nanocapsules of the present invention areadaptable for the controlled release of a variety of encapsulatedtherapeutic agents, including macromolecules, into in vivo circulationof a recipient upon administration thereto. The nanocapsule compositionsof the present invention are further adapted to encapsulatetherapeutically effective concentrations of an agent of therapeuticinterest and deliver the same into in vivo circulation of a recipient.

In addition, the nanocapsules of the present invention are adaptable toprovide controlled release of an encapsulated agent of therapeuticinterest in vivo.

According to one embodiment of the present invention a method forpreparing a biodegradable nanocapsule having a circulation half-time ofat least 35 hours in vivo is provided. This nanocapsule has been shownto effectively deliver an exemplary macromolecule of interest into invivo circulation with a controlled release rate providing a circulationhalf-time of the macromolecule of at least 14 hours.

A number of novel approaches were employed in accordance with thepresent invention to adapt the nanocapsule compositions to releaseencapsulated macromolecules at controlled rates in vivo. For example, ananocapsule composition of the present invention was adapted to alterthe release rate of encapsulated proteins from a half time of 2 hours instep-wise fashion to a release rate of a half time of at least 24 hours.Accordingly, the present invention provides a nanocapsule compositionhaving an improved circulation time that can maintain an encapsulatedagent of therapeutic interest in in vivo circulation for a prolongedperiod of time. As such, the nanocapsule of the present inventionprovides a versatile carrier for in vivo delivery and controlled releaseof a plurality of agents of therapeutic interest encapsulated therein.

One aim of the present invention is to provide a multi-purposebiodegradable nanocapsule having an improved in vivo circulation time.

Another aim of the present invention is to provide a biodegradablenanocapsule membrane having an effective circulation half-time of atleast 35 hours.

Another aim of the present invention is to provide a biodegradablenanocapsule composition having an effective circulation half-time of atleast 6 hours.

Another aim of the present invention is to provide a biodegradablenanocapsule composition having an effective circulation half-time of atleast 14 hours.

Yet another aim of the present invention is to provide a biodegradablenanocapsule composition having an effective circulation half-time of atleast 24 hours.

Another aim of the present invention is to provide a multi-purposebiodegradable nanocapsule that is selectively permeable to biologicalagents of therapeutic interest.

Another aim of the present invention is to provide a multi-purposebiodegradable nanocapsule composition adaptable for the controlledrelease or delivery of a variety of therapeutic agents of interest invivo.

A further aim of the present invention is to provide a method forpreparing a nanocapsule composition having an improved circulation timein vivo.

Yet a further aim of the present invention is to provide a method fordelivering an agent of therapeutic interest in vivo.

In accordance with the present invention there is provided abiodegradable polymeric nanocapsule membrane composition adaptable forencapsulating an agent of therapeutic interest and enhancing in vivocirculation time thereof, said nanocapsule membrane compositioncomprising a copolymer of polylactic acid polymer and polyethyleneglycol wherein said copolymer is soluble in acetone and insoluble inwater.

In accordance with another aspect of the present invention there isprovided a hemoglobin nanocapsule composition, said compositioncomprising a biodegradable polymeric nanocapsule membrane encapsulatinga therapeutically effective concentration of a hemoglobin preparation;said nanocapsule membrane comprising a copolymer of polylactic acidpolymer and polyethylene glycol; said copolymer being soluble in acetoneand insoluble in water; wherein said nanocapsule composition isadaptable for enhancing the in vivo circulation time of said hemoglobinpreparation.

The hemoglobin nanocapsule composition of the present invention mayinclude other biological agents known to inhibit the production ofmethemoglobin. Furthermore, the hemoglobin nanocapsule composition ofthe present invention may be adapted to be selectively permeable tomolecules present in in vivo circulation, that prevent the hemoglobin inthe nanocapsules from becoming methemoglobin.

In accordance with another aspect of the present invention there isfurther provided a method for preparing a nanocapsule composition havingan enhanced circulation time in vivo, said method comprising: (a)preparing a copolymer mixture of a polylactic acid polymer andpolyethylene glycol (PLA-PEG); (b) heating said copolymer mixture; (c)adding an aqueous solution comprising an agent of therapeutic interestto said copolymer mixture; (d) precipitating said copolymer mixture fromsaid aqueous solution; and (e) extracting the nanocapsule compositiontherefrom; wherein said composition comprises a biodegradable, polymericnanocapsule membrane encapsulating said agent of therapeutic interest.

The method of the present invention for preparing a nanocapsulecomposition having enhanced circulation time in vivo, may furtherinclude incubating the agent of therapeutic interest with a cross-linkercomponent prior to mixing the agent together with the PLA-PEGpreparation. Alternatively, or in addition, the method for preparing ananocapsule composition of the present invention may further includecross-linking at least some of the encapsulated agent of therapeuticinterest to an internal surface of the nanocapsule, wherein across-linker component is added subsequent to the mixing of the PLA-PEGmixture with the agent of therapeutic interest.

A cross-linker component of the present invention may be glutaraldehyde.However, it is fully contemplated that alternatives to this cross-linkercomponent, as known in the art, may be employed.

In accordance with still a further aspect of the present invention thereis further still provided a delivery system for enhancing thecirculation time of an agent of therapeutic interest in vivo, saidsystem comprising: a biodegradable polymeric nanocapsule compositioncomprising of a copolymer membrane encapsulating said agent oftherapeutic interest; wherein said copolymer membrane includes acopolymer of polylactic acid and polyethylene glycol and is soluble inacetone and insoluble in water; said copolymer membrane being adaptableto deliver the encapsulated agent of therapeutic interest into in vivocirculation at a controlled rate of release.

In accordance with the present invention there is still further provideda delivery system for providing the step-wise release of an agent oftherapeutic interest in vivo, said system comprising: a plurality ofbiodegradable polymeric nanocapsule compositions each adapted to releasean encapsulated agent of therapeutic interest at a differentpredetermined rate of release in vivo; wherein each of said plurality ofbiodegradable polymeric nanocapsules includes a copolymer membranecomprising a copolymer of polylactic acid and polyethylene glycol, saidcopolymer being soluble in acetone and insoluble in water.

Furthermore, the drug delivery system of the present invention may befurther adapted to be selectively permeable to biological componentspresent in in vivo circulation of the recipient. According to thisembodiment of the present invention, the quality and integrity of theencapsulated agent of therapeutic interest may be maintained.

Alternatively, or in addition to, the drug delivery system of thepresent invention may also be adapted to encapsulate other biologicalcomponents together with the agent of therapeutic interest. According toan alternative embodiment of the present invention, a drug deliverysystem having improved in vivo stability is provided whereby at least aportion of the encapsulated drug or agent of therapeutic interest iscross-linked to the internal surface of the nanocapsule.

In accordance with the present invention there is also provided ananocapsule composition comprising a biodegradable polymeric nanocapsulemembrane encapsulating a therapeutically effective concentration of amacromolecule; said nanocapsule membrane comprising a copolymer ofpolylactic acid polymer and polyethylene glycol; said copolymer beingsoluble in acetone and insoluble in water; wherein said nanocapsulecomposition is adaptable for enhancing the in vivo circulation time ofsaid macromolecule.

For the purpose of the present invention the following terms are definedbelow.

The term “therapeutic agent” is intended to mean any agent having atherapeutic potential when administered in vivo, including, withoutlimitation, macromolecules such as hemoglobin, proteins, enzymes, RNA,DNA, and genes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a graphical representation of the circulation timesof a variety of nanocapsule compositions in accordance with the presentinvention;

FIG. 2 illustrates a graphical representation of the retention times ofHb in a variety of nanocapsule compositions in accordance with thepresent invention;

FIGS. 3A and 3B illustrate the circulation time of a variety ofnanocapsule compositions in accordance with the present invention;

FIG. 4 illustrate the circulation time of a variety of nanocapsulecompositions in accordance with the present invention; and

FIG. 5 illustrates a graphical representation of the concentration ofnon-RBC Hb (g/dl) over time.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, improved biodegradablenanocapsules having an increased in vivo circulation time are provided.Preferably, the biodegradable nanocapsules of the present invention arepolymeric nanocapsules. The biodegradable nanocapsules of the presentinvention are adaptable to encapsulate agents of therapeutic interestand deliver the same into in vivo circulation upon administration to arecipient. Furthermore, the nanocapsule compositions of the presentinvention are adaptable for prolonging the in vivo circulation time ofthe encapsulated agent contained therein. For practical therapeuticapplications, such as blood substitutes, a nanocapsule compositionhaving an in vivo circulation half-time of at least 14 hours, ispreferred. As a result, extensive research and development was carriedout in connection with the present invention to devise a nanocapsulecomposition having an increased circulation time in vivo. A novelapproach is herein demonstrated, without limitation, in connection withhemoglobin to provide a hemoglobin nanocapsule composition having animproved circulation time and/or controlled rate of release, in vivo.

The present invention provides a novel nanocapsule composition adaptableto encapsulate and effectively deliver agents of therapeutic interest.For example, together with hemoglobin, the nanocapsule composition maybe employed to provide a blood substitute. By increasing the in vivocirculation time of an encapsulated agent, a nanocapsule composition ofthe present invention can significantly increase the duration offunctioning of the encapsulated agent in vivo, thereby enhancing thetherapeutic effect of the composition. In addition, the nanocapsulecomposition of the present invention can effectively deliver a givendosage of a therapeutic agent in a controlled manner, thus improving theeffectiveness of the dosage while reducing the overall administrationrequired.

According to a preferred aspect of the present invention, atherapeutically effective amount of hemoglobin is encapsulated in ananocapsule composition and adapted for delivery into the in vivocirculation of a recipient, administered therewith. The encapsulatedhemoglobin carries out its function as a blood substitute intransporting oxygen in vivo. Furthermore, when the encapsulatedhemoglobin is released from the nanocapsule membrane, it continues tofunction in the transport of oxygen, thus serving to enhance the totallength of function of ht encapsulated hemoglobin in vivo.

The present invention further provides a novel delivery system for thecontrolled release of a therapeutic agent into in vivo circulation. Theability to modify or adapt the nanocapsule compositions of the presentinvention to provide a controlled rate of release of the encapsulatedagent serves to further provide a novel therapeutic delivery system.Furthermore, a variety of nanocapsule compositions of the presentinvention may be employed together to provide an effective step-wisetherapeutic delivery system. According to this aspect of the presentinvention, each nanocapsule composition is adapted to deliver anencapsulated agent of therapeutic interest at a predetermined rate ofrelease. When a plurality of these nanocapsule compositions, each havinga predetermined rate of release, are administered simultaneously, theencapsulated agent of therapeutic interest is released in vivo in astep-wise fashion.

According to one embodiment of the present invention biodegradablepolymeric nanocapsules are provided having an in vivo circulation halftime of at least 35 hours, as discussed below. The nanocapsulecompositions of the presents invention are adapted to encapsulate avariety of different therapeutic agents, including macromolecules suchas hemoglobin, enzymes, RNA, DNA, and other proteins including verylarge polymerized hemoglobin and enzymes and deliver the same in vivo ata controlled rate of release thereby improving the in vivo circulationtime of the encapsulated agent. The nanocapsules compositions of thepresent invention are further adapted to serve as carriers fortherapeutically effective concentrations of an agent of therapeuticinterest, providing the controlled in vivo release thereof. According tothe present invention, an agent of therapeutic interest may be, withoutlimitation, a macromolecule.

According to one embodiment of the present invention, while thenanocapsules maintain their circulation half-time of 35 hours, we wereable to vary the release rate of the enclosed proteins from a releaserate of a half time of 2 hours in step-wise fashion to a release rate ofa half-time of at least 24 hours in rats. As discussed furtherhereinbelow, it is expected that the in vivo circulation half-time ofthe nanocapsule compositions of the present invention would be evengreater in humans. Thus, according to the present invention a versatiledelivery system for the controlled in vivo release of a variety oftherapeutic agents is provided.

An agent of therapeutic interest, for example, may be encapsulated by ananocapsule composition of the present invention and introduced into invivo circulation of a recipient by any one of a variety of methods ofnanocapsule delivery. For example, a nanocapsule composition of thepresent invention may be administered by, without limitation,intravenous, intramuscular, intraperitoneal, or subcutaneous delivery,orally or by topical administration. The nanocapsule composition can bemodified to provide a controlled rate of release of the encapsulatedagent from the nanocapsule carrier. As a result, the agent of interestmay be introduced into in vivo circulation at a controlled rate ofrelease for achieving a desired therapeutic effect. The rate of releaseof a nanocapsule composition of the present invention can be adapted toachieve a desired rate of release of an encapsulated agent oftherapeutic interest in a variety of ways, as exemplified herein below.

As exemplified in accordance with the present invention, it is possibleto encapsulate hemoglobin within a nanocapsule carrier to obtain atherapeutically effective circulation time thereof, in vivo. Thus, it isfully contemplated that the nanocapsule composition of the presentinvention is adaptable to encapsulate virtually any agent of therapeuticinterest including macromolecules, in a therapeutically effectiveconcentration, for the purpose of providing a controlled drug deliverysystem.

In accordance with the present invention, a method for the preparationof an nanocapsule composition may include, without limitation, alteringthe permeability of a nanocapsule membrane; decreasing the rate ofdegradability of the nanocapsule membrane; or increasing the molecularsize of the enclosed agent of therapeutic interest to adapted thenanocapsule composition to maintain a desired circulation half-timeand/or controlled rate of release of the encapsulated agent oftherapeutic interest is provided.

Materials & Methods

Materials

Polylactic Acid

Polylactic acid (PLA) is obtained from Polysciences Inc. (Canada).Isobutyl 2-cyanoacrylate (IBCA), surfactants (Tween 20™, Span 85™,Triton X 100™, and Pluronic F68™), ethyl cellulose andL-α-Phosphatidylcholine (hydrogenated) and other phospholipids, such asdistearoyl phosphatidylcholine (DSPC) or DSPG and tocopherol acetatewere obtained from Sigma Chemical Co. (U.S.A.). Dialysis membrane(Spectrapor 5™) is purchased from Fisher Scientific Co. Diethyl ether,ethyl acetate, cyclohexane, and chloroform are purchased from BDHChemical (Canada). All the other chemicals are of reagent grade, forexample, methoxypolyethylene glycol (MW 2000, MW 3350) andstannous-2-ethylhexanoate.

Methods

Preparation of Hemoglobin Solution

Stroma free hemoglobin is prepared according to the standard method(Chang, T. M. S., 1997, “Blood Substitutes:Principles, methods, productsand clinical trial”, vol. I, Karser publisher, Basel). Briefly,hemoglobin is obtained by hypotonic hemolysis of bovine red cells and itis made stroma-free by toluene extraction and is clarified by high speedcentrifugation. The resulting solution contained 10 to 15 ghemoglobin/dl. In order to minimize the formation of methemoglobin, themanipulation is carried out at 4° C. and the hemoglobin solution iscontrolled at pH 7.4.

Preparation of Biodegradable Polymer Nanocapsule Compositions (StandardMethod)

Organic Phase:

Dissolve 100 mg (d.l)-polylactic acid (MW 25000) (Polysciences,Warrington, Pa.) in 8 ml acetone. Dissolve 50 mg hydrogenated soybeanphosphatidylcholine (Avanti Polar Lipids, Alabaster, Ala.) in 4 mlethanol with help of Ultrasonic water bath (very low power). Above twosolutions were mixed (add either one to another), and use as organicphase.

Aqueous Phase:

Take 0.04 ml of Tween 20, mix with 25 ml 15% (g/dl) hemoglobin.

Preparation:

Slowly inject (8 ml/min.) the organic phase into the aqueous phase undermagnetic stirring [with Therm-O-Swirl Stirrer (Precision Scientific Co.,Chicago) setting to 6)], under 4° C. The injection head was made with a0.2 ml pipette tip. The nanocapsules were formed immediately, but, thesuspension was keeping stirring for 15 min. The suspension prepared is37 ml. The organic solvent was partly removed from above suspensionprepared by rotary evaporator under vacuum at 20° C. for about 10minutes. The suspension obtained was 33 ml (i.e. removed 4 ml organicsolvent). The remaining suspension was mixed with 15 ml of 0.9% NaCl.Then the organic solvent and free hemoglobin were removed byultrafiltration (by Amicon ZM 500,000 membrane, MW cut off 500,000). Thesuspension was repeatedly washed by 0.9% NaCl by ultrafiltration. Theoperation was carried out at 4° C., with nitrogen.

The nanocapsule compositions prepared according to the standardprocedures outlined above were adapted, as described hereinbelow, toprovide the nanocapsule compositions of the present invention havingimproved circulation time and controlled release rates.

As exemplified in accordance with the examples provided hereinbelow,Example II outlines the preparation of nanocapsules having a circulationhalf-time of 35 hours. In this example, although the nanocapsulesthemselves circulate for 35 hours T1/2 the contents were found to leakout with a T1/2 of 2 hours or less. Example III exemplifiesmodifications to the membrane characteristics of Example II whichresults in nanocapsules that can retain encapsulated agents for longerperiods of time. Example IV exemplifies further modifications resultingin improved retention times of encapsulated agents. Example IV combines(1) the methods of modifications of the nanocapsule membrane fromExamples II & III with (2) modification to the encapsulated agentsthemselves e.g. cross-linking macromolecules by different degrees ofpolymerisation to provide larger molecular weights prior toencapsulation. The details of the modifications are described for eachof the examples. Each example from II to IV has a number of differentmodifications and approaches.

The present invention will be more readily understood by referring tothe following examples which are given to illustrate the inventionrather than to limit its scope.

EXAMPLE I Standard Polymeric Nanocapsule Preparations

We first varied the membrane compositions of 5 variations of polymericnanocapsules and studied their effects on circulation time.

(1) Polylactic acid(PLA)-nanocapsules were prepared by as describedabove for standard nanocapsules. These PLA-nanocapsules displayed acirculation half-time of about two hours (FIG. 1).

(2) The addition of phospholipids to the polymer membrane ofnanocapsules of the standard procedure resulted in only modest increasesin circulation time (this item not shown in FIG. 1). In this case,hydrogenated soybean lecithin (HSPC) or distearoyl phosphatidylcholine(DSPC) was incorporated into the PLA nanocapsules.

(3) The incorporation of lipid-polyethylene glycol (PEG) to the polymermembrane of nanocapsules did not result in any significant increases inthe circulation time (FIG. 1—PLA-PEG-lipid). Here,1,2-dietearoyl-glycero-3-phophoethanolamine-N-[poly(ethyleneglycol)-2000] was incorporated into the membranes of the PLAnanocapsules.

(4) Adsorption of PEG to the nanocapsules was also performed. 7% PEG (MW15000) was added to the PLA nanocapsules and left in the suspension for6 hours. Unfortunately, there were no significant increases incirculation time (FIG. 1—PEG adsorbed to PLA).

(5) Standard PLA-PEG copolymers were prepared by mixing 10 g ofmethoxypolyethylene glycol (MW 2000) and 10 g of DL-lactic acid andstirred under nitrogen at 160° C. Then 50 μl of stannous2-ethylhexanoate was added. The mixture was kept at 160° C. for 3 hours.However, this polymer was soluble in water and therefore could not beused in the Hb PLA-nanocapsules process. This composition was deemedunsuitable for use in the encapsulation of water soluble macromoleculeswithin nanocapsules.

EXAMPLE II Preparation of Hemoglobin Encapsulated NanocapsuleCompositions

-   (1) Organic Phase:

Dissolve 100 mg of the PLA-PEG copolymer prepared as described indetails below (Method 2) in 8 ml acetone. Dissolve 50 mg hydrogenatedsoybean phosphatidylcholine (Avanti Polar Lipids, Alabaster, Ala.) in 4ml ethanol with the help of Ultrasonic water bath (very low power).Above two solutions were mixed and use as organic phase.

-   (2) Aqueous Phase:

Take 0.04 ml of Tween 20, mix with 25 ml 15% (g/dl) hemoglobin.

-   (3) Preparation:

Slowly inject (8 ml/min.)(The injection head is made with a 0.2 mlpipette tips) the organic phase into the aqueous phase under magneticstirring [with Therm-O-Swirl Stirrer (Precision Scientific Co., Chicago)setting to 6)], at 4° C. The nanocapsules were formed immediately, but,the suspension was keeping stirring for 15 min. The suspension preparedis 37 ml.

The organic solvent was partly removed from above suspension prepared byrotary evaporator under vacuum at 20° C. for about 10 minutes. Thesuspension obtained was 33 ml (i.e. removed 4 ml organic solvent). Theremained suspension was mixed with 15 ml of 0.9% NaCl. Then the organicsolvent and free hemoglobin were removed by ultrafiltration (by AmiconZM 500,000 membrane, MW cut off 500,000). The suspension was repeatedlywashed by 0.9% NaCl by ultrafiltration.

The operation was carried out at 4° C., with nitrogen.

It was subsequently determined that a suitable nanocapsule polymer wouldpreferably be soluble in acetone and insoluble in water. The followingnovel copolymers were investigated for use in preparing nanocapsules ofthe present invention:

Method 1—One gram of D,L-PLA[M.W. 10,000] and 0.5 g of PEG [M.W. 3350]were dried under vacuums overnight. 5 ml of acetone was added. Themixture was heated to 100° C. for 1 hr under nitrogen. After adding 20μl of stannous-2-ethylhexanoate, the mixture was heated to 180° C. foranother 6 hrs under nitrogen. The final polymer is soluble in acetone.This copolymer was subsequently employed with hemoglobin in thepreparation of a biodegradable nanocapsule composition as describedabove. However, this PLA-PEG copolymer preparation (PEG-PLA method 1)did not increase the circulation half-time sufficiently (FIG. 1).

Method 2—One and a half grams of DL-PLA [M.W. 10,000] and 0.75 g ofmethoxypolyethylene glycol [M.W. 2000] were dried under vacuumsovernight. The mixture was heated to 180° C. for 2 hr under nitrogen.After adding 10 μl of stannous-2-ethylhexanoate, the mixture was heatedat 180° C. for another 3 hours under nitrogen. The final polymer issoluble in acetone. This copolymer was subsequently employed withhemoglobin in the preparation of a biodegradable nanocapsule compositionas described above. We determined circulation half-times using thispreparation (PLA-PEG method 2) in rats and found a circulation half-timeof 35 hours (FIG. 1). Thus, this PLA-PEG copolymer preparation wasidentified as a novel candidate for an improved biodegradable polymericnanocapsule.

Method 3—Another study was carried out to determine if the use of ahigher MW PEG would further increase the circulation time. Two grams ofD,L-PLA [M.W. 10,000] and 1 g of PEG [M.W. 3350] were dried undervacuums overnight. 5 ml of acetone was added. The mixture was heated to100° C. for 1 hr under nitrogen. Then, the mixture was heated to 180° C.for another 10 hours under nitrogen. This copolymer is soluble inacetone. This copolymer was subsequently employed with hemoglobin in thepreparation of a biodegradable nanocapsule composition as describedabove. This preparation did not result in a sufficient increase incirculation half-time (FIG. 1).

EXAMPLE III Use of PLA-PEG Copolymer as a Nanocapsule Carrier

Once we obtained a nanocapsule with sufficient circulation half-time, weproceeded to study this nanocapsule preparation with the exemplarymacromolecule, hemoglobin (Hb). In light of the 35 hour circulationhalf-time obtained with the PLA-PEG copolymer preparation (Method 2)above, this preparation was further studied as a nanocapsule membranecarrier for the in vivo delivery of hemoglobin. According to the presentinvention, hemoglobin is employed as an exemplary macromolecule toillustrate the properties of the nanocapsule of the present invention asa carrier for therapeutic agents of interest. However, the presentinvention is not limited thereto.

Hemoglobin nanocapsules were successfully prepared (as described above).

These nanocapsules displayed circulation half-time of about 35hours—similar to those nanocapsules prepared without Hb. However, theencapsulated hemoglobin was found to leak out of the nanocapsules afterinfusion into in vivo circulation and rapidly disappear. The circulationhalf-time of the Hb component within the nanocapsule carrier was foundto be less than 2 hours, while, the nanocapsules themselves continue tocirculate with a half-time of 35 hours—even though the membrane isslowly degraded and becomes leaky.

Various steps were employed to solve the problem of leakage of amacromolecule component from the nanocapsules of the present invention,as outlined in Table 1. The hemoglobin nanocapsules were prepared usingthe detailed method described for PLA-PEG nanocapsules. After this, theresulting hemoglobin nanocapsules were cross-linked usingglutaraldehyde. The purpose of this was to cross-link the hemoglobininside the nanocapsules to provide large macromolecules, e.g.polyhemoglobin. Thus, decreasing the rate of their release from thenanocapsules. In addition, hemoglobin molecules were cross-linked nearthe internal surface of the nanocapsules so as to decrease thepermeability of the membrane of the nanocapsules. Table 1 shows thevariations employed to adapt the release rate of the nanocapsulecompositions of the present invention, as exemplified together withhemoglobin, including (1) the amount of glutaraldehyde used as shown bycross-linker/hemoglobin ratio; (2) the duration of the cross-linkingprocess as shown by cross-link time; and (3) the concentration ofglutaraldehyde used. The circulation half-times of the resulting HbPLA-PEG nanocapsules are shown in the column under T1/2.

TABLE 1 Procedures used to effect an increase in emoglobin retentiontime within PLA-PEG-nanocapsules Crosslinker/Hb Crosslink (Xlk/Hb) TimeCrosslinkConc. PROCEDURE ratio (Xlkt) (Xlkc) T_(1/2) Hb-nanocapsule  1.3h before cross-linking 980914 Hb-nanocapsule 8:1  2 h  0.5 M  4.0 981027Hb-nanocapsule 8:1  2 h  0.5 M  6.5 990119 Hb-nanocapsule 16:1   3 h0.25 M 10.0 990126 Hb-nanocapsule 6:1  5 h 0.25 M 14.0 990202C2Hb-nanocapsule 16:1   5 h  0.5 m 16.0 990302 Hb-nanocapsule 16:1  20 h 0.5 M 12.4 h 990304

Increasing the cross-linking time to 5 hours with cross-linkerconcentrations of 0.5M increases the circulation half-time to 16.0 hours(FIG. 2). This is a vast improvement compared to the 2 hours for Hbnanocapsules that have not been cross-linked. This is also higher thanthe 8 to 12 hours for cross-linked hemoglobin solutions in rats.Furthermore, given that circulation half time is usually much lower inrats than in humans, this result suggests that significant improvementin circulation time can be achieved in human, with the nanocapsulecompositions of the present invention. However, we continued to devisenovel approaches to decrease even further the release rates ofhemoglobin from this nanocapsule composition after infusion.

Cross-Linking Procedures for Hb Nanocapsules

To strengthen the Hb nanocapsule membrane, different approachesincluding cross-linking were employed. A summary of the differentmethods studied is shown in Table 2 below and illustrated in FIGS. 3Aand 3B.

Table 2 outlines further studies employed in investigating nanocapsulestrength, using the above approach of cross-linking the PLA-PEGhemoglobin nanocapsules as shown in Table 1. As presented in Table 2,“Xlink” refers to the ration of glutaraldehyde and hemoglobin as inTable 1; “PLA-PEG” refers to the molecular weight of PLA/PEG used forthe preparation of the PLA-PEG hemoglobin nanocapsules; “React” refersto the time (hrs) of cross-linking with the cross-linker; “Separation”refers to the step of concentration of the resulting nanocapsules asdetailed in the detailed method for the preparation of PLA-PEGhemoglobin nanocapsules described earlier; “Ethanol” refers to the useof ethanol in the preparation. The first column of Table 2 provides asummary of columns 2 to 6. e.g.

5KX(12:1) 0.25F refers to: PLA with molecular weight of 15 Kd (xlinkerratio of 12:1) reaction time of 0.25 and filtration for separation.

TABLE 2 React Etha- METHODS Xlink (PLA-PEG) (hrs) Separation nol 5K5000/2000 filtration 5KX(12:1)0.25F 12:1  5000/2000 0.25 filtration5KX(8:1)2.5F 8:1 5000/2000 2.5 filtration 5KX(27:1)0.1F 27:1  5000/20000.1 filtration 5KX(27:1)0.1F-1 27:1  5000/2000 0.1 filtration5KX(36:1)5F 36:1  5000/2000 5 filtration 5KX(36:1)5F-1 36:1  5000/2000 5filtration 5KX(36:1)5F-2 36:1  5000/2000 5 filtration 5KX(16:1)5F 16:1 5000/2000 5 filtration 5KX(16:1)20F 16:1  5000/2000 20 filtration5KX(8:1)5F 8:1 5000/2000 5 filtration 5KX(8:1)20F 8:1 5000/2000 20filtration 5KX(8:1)20F-1 8:1 5000/2000 20 filtration 5KX(8:1)20G-2 8:15000/2000 20 Gel-Dialysis 5KX(8:1)20G 8:1 5000/2000 24 Gel-Dialysis5KX(8:1)21GA30 8:1 5000/2000 21 Gel-Dialysis 30% 5KX(8:1)20GA25 8:15000/2000 20 Gel-Dialysis 25% 5KX(8:1)20GA20 8:1 5000/2000 20Gel-Dialysis 20% 15KX(8:1)24G 8:1 15000/2000  24 Gel-Dialysis15KX(8:1)48G 8:1 15000/2000  48 Gel-Dialysis 15KX(8:1)48G-1 8:115000/2000  48 Gel-Dialysis 15KX(8:1)48G-2 8:1 15000/2000  48Gel-Dialysis 15KPOLYHb PolyHb 15000/2000  PolyHb Gel-DialysisCirculation Time of Hb Nanocapsules

Total blood volume in a 200 gram rat is about 12 ml. 30% top-loading isthe infusion of 30% of the total blood volume of a rat with ananocapsule suspension, i.e. 3.5 ml, into the rat. Results oftop-loading of 30% are shown in FIGS. 3A and 3B. FIGS. 3A and 3Billustrate the circulation time obtained from those methods of Table 2with the best results obtained using PLA of MW of 5K-15K in thenanocapsule composition.

As illustrated in FIGS. 3A and 3B, Hb nanocapsules circulate well in thefirst 6 hours. However, when samples are taken 24 hours after infusion,they are down to 15-25%. The T_(1/2) of the nanocapsule compositionimproved from the original (5K) of less than 2 hours to around 14-16hours.

Biodegradability

Dialysis-gel absorption method was used to separate free Hb or freecross-linked Hb from the PLA-PEG hemoglobin nanocapsules. When Hbnanocapsules samples were obtained 2 hours after infusion and placed inthis system, no free Hb was extracted showing that the Hb nanocapsulesremained intact. When Hb nanocapsules were incubated with plasma in thedialysis tubing—no Hb was extracted until after 6 hours. After this,free Hb slowly appeared in the suspension and was extracted into thegel, showing that there is slow breakdown in the integrity of the PLAmembrane due to biodegradation in plasma starting after 6 hours.

Polymer Composition

In view of the fact that the larger the MW of PLA, the slower is thebiodegradability, PLA with a MW of 15-25K was used to form the PEG-PLAcopolymer preparation. The use of a higher MW PLA (15K-25K) requires theuse of 200° C. for the copolymerisation procedure for PEG-PLA, which mayresult in the breakdown of the PLA molecule. Size exclusionchromatograph with respect to polystyrene standard, M_(n)=6900,M_(w)=8600, M_(z)=7000, M_(w)/M_(n)=1.24 shows that the molecular weightof PLA-PEG copolymer was decreased to only 6,900. Thus, substantialbreakdown of the PLA molecule was observed.

Results obtained with PLA having a MW of 15-25K are exemplified in FIGS.4A and 4B. No improvement in circulation time was observed, except inthe case of 15 POLYHb as discussed hereinbelow.

EXAMPLE IV Use of PLA-PEG Copolymer Nanocapsule with Polyhemoglobin

Polyhemoglobin is formed by polymerizing hemoglobin molecules to providemacromolecules that are larger in size. Preferably, each polyhemoglobincontains 4-5 hemoglobin molecules chemically linked together.

We have shown that the particle counts of PEG-PLA nanocapsules (bothempty and Hb containing) have a circulation half time of 35 hours.However, the circulation T_(1/2) of Hb in the Hb nanocapsules is lowerbecause of the leakage of the enclosed Hb with time. Thus, we haveinvestigated the use of encapsulating polyHb with a MW of about 240K to360K. This will have less problem of leakage than the smaller native Hbof about MW 64K. As shown in FIGS. 4A & 4B with respect to 15 KPOLYHB,this significantly increased the T_(1/2) from the original 1-2 hours to14 hours. Accordingly, the present invention provides methods forimproving the circulation time of nanocapsule compositions comprisingencapsulated agents of therapeutic interest. For example, amacromolecule can be cross-linked to form larger molecules andsubsequently be encapsulated by a nanocapsule composition of the presentinvention. Furthermore, other macromolecules of interest may be mixedwith hemoglobin to form a polyhemoglobin complex.

According to the present invention, cross-linking of an agent oftherapeutic interest may be performed prior to, or after encapsulation.In addition, encapsulated agents may also be cross-linked to an interiorsurface of the nanocapsule membrane.

(1) Use of Polyhemoglobin Containing Less Single Hemoglobin Molecules

Variations in membrane composition were investigated to improve thecirculation time of the nanocapsules and retention of the hemoglobininside the nanocapsules during circulation. As a result, effortsfocussed on preparations that will maintain a given hemoglobin level totransport oxygen after infusion. This is achieved by improving the Hbnanocapsules according to the methods described above by decreasing thebiodegradability of the nanocapsule, altering the permeability of thenanocapsule, such as by cross-linking some of the encapsulated agent tothe membrane with a cross-linking agent and/or by using a largermacromolecules for encapsulation. As a result, nanocapsule compositionsof the present invention is adapted to provide: (1) a higher initialconcentration of an encapsulated agent in vivo and (2) increase thecirculation time of the encapsulated agent.

In previous studies, the slope of the disappearing curve of thehemoglobin concentration was analyzed and extrapolated to zero tocalculate the circulation half-time. This method is acceptable forcomparing and screening a large number of Hb nanocapsules prepared bydifferent formulations. However, this type of analysis is more suitablefor looking at the rate of release of drug delivery systems. Inaccordance with one aspect of the present invention, the actual amountof hemoglobin that is circulating to supply oxygen was investigated.Accordingly, the actual level of hemoglobin remaining in the circulationrather than the slope of the curve was determined.

Using a blood volume in rats of about 60 ml/kg, and knowing the amountof Hb nanocapsules infused, the maximal level of hemoglobin possibleafter each infusion was calculated. After this the concentration ofhemoglobin in the circulation was followed. Circulation half-time inrats for Hb blood substitutes is known to be much shorter than in human.Thus, a baseline reference in comparing the significance of resultsobtained in rats to human was made. Simultaneous studies usinggluataraldehyde PolyHb were conducted. Knowing that the circulation halftime of these preparations in human is about 24 hours, we obtained adirect basis for extrapolation to human. However, rats are known to havea much more avid reticulo-endothelial system (RES) for the removal ofparticulates like nanocapsules. Thus, when extrapolated to human for Hbnanocapsules the circulation half-time would be even higher than what itwould actually be in the rats.

(2) Baseline Studies Using Polyhemoglobin

In order to have valid comparisons, all studies in rats used the sameprotocol. Polyhemoglobin and Hb nanocapsules suspensions were adjustedto have the same hemoglobin concentration of 10 gm/dl. The volumeinfused was the same for both PolyHb and Hb nanocapsules and is 30% ofthe total blood volume (30% top-load).

Table 3 shows that 30% top-load using preparations with Hb (10 gm/dl) inrats resulted inpolyhemoglobin (17:1): maximal non-rbc Hb conc. 3.35gm/dl, falling to half its maximal concentration of 1.67 gm/dl in 14hrs. From here on, PolyHb (17:1) was used as the basis for comparison toall other preparations including the time for the circulating non-rbc Hbof different preparations to reach 1.67 gm/dl. Thus, in the case ofpolyhemoglobin (10:1): maximal non-rbc Hb conc is 3.10 gm/dl, falling to1.67 gm/dl in 10.4 hrs.

30% top-load using EncHb(10:1) resulted in a maximal non-rbc Hb level ofonly 3.05 gm/dl (S.D.=0.03) (Table 3). This refers to hemoglobin thathas been first cross-linked with glutaraldehyde with acrosslinker:hemgolobin ratio of 10:1 and then encapsulated withinPLA-PEG nanocapsules. The non-rbc Hb falls to 1.67 gm/dl in 12.3 hoursin rats (21 hours in human equivalent).

Calculations based on body weight, blood volume, plasma volume anddilution factors illustrate that the maximum non-rbc hemoglobinconcentration for Hb nanocapsules is at least 3.6 gm/dl rather than only3.05 gm/dl as for EncHb(10:1) in Table 3. This seems to show that asignificant part (about 16%) of the infused Hb nanocapsules was removednearly immediately on infusion. Thus the next step is to try to preventthis.

In the above preparation, hemoglobin was first cross-linked intopolyhemoglobin before being encapsulated into the nanocapsules.

The larger molecular size of polyhemoglobin delays the leakage of thehemoglobin as the nanocapsule membrane degrades in the circulation, andtherefore increase the circulation half-time (Table 3). We carried out amore detailed analysis of the molecular weight distribution of thepolyhemoglobin used in the above preparation based on PolyHb(10:1). Wethen used a higher degree of polymerization as described in the examplesbelow to improve the degree of polymerization to markedly reduce theamount of single tetrameric hemoglobin (PolyHb 17:1) and encapsulationwas performed.

(i) Formulation “EncapHb (17:1)”

The polyhemoglobin “EncapHb (17:1)” was used in the followingformulation for hemoglobin nanocapsules. The ratio of the cross-linker(glutaraldehyde) to hemoglobin used in the cross-linking step forforming polyhemoglobin was 17:1 for this formulation. 100 mg ofPLA-coPEG and 50 mg phospholipid were dissolved in a mixed solution ofethanol (3 ml) and acetone (6 ml). Then this solution was slowlyinjected into 25 ml of the above polyhemoglobin solution containing0.24% Tween 20 under constant magnetic stirring. Diffusion of ethanoland acetone into the aqueous phase resulted in particle formation. Theethanol and acetone in the aqueous phase was easily eliminated bydialysis against saline solution at 4° C.

TABLE 3 time to Formula max Hb 1.67 g/dl (hrs) PolyHb (17:1): 3.35 gm/dl14.0 (rats)   24 (Human) PolyHb (10:1): 3.10 gm/dl 10.4 (rats)   17(Human) EncHb (10:1): 3.05 gm/dl 12.3 (rats)   21 (Human) EncHb (17:1):3.58 gm/dl 17.1 (rats)   29 (Human) EncHb 1.5 (5 k): 3.60 gm/dl 20.0(rats)   34 (Human) EncHb 1.0 (5 k)XL 3.60 gm/dl 20.3 (rats)   35(Human) EncHb 1.0 (15 k): 3.57 gm/dl 21.2 (rats)   36 (Human) EncHb 1.5(15 k): 3.65 gm/dl 23.3 (rats) 39.9 (Human EncHb 1.5 (15 k)XL 3.66 gm/dl24.2 (rats) 41.5 (Human)

As shown in Table 3, two minutes after infusion, the maximal non-rbc Hbwas 3.58 gm/dl(S.D.=0.04). This is significantly higher than the 3.05gm/dl (S.D.=0.04) for the earlier Hb nanocapsules (EncHb (10:1). Thisalso approaches the maximal possible initial non-rbc Hb concentration.Furthermore, the slope of the disappearance is also much slower, butwhat is more important is that it took 17.1 hrs (rats) or 29.3 hrs(human) for the non-rbc Hb level to fall to 1.67 gm/dl as compared to12.3 hrs (rats) or 21.1 hrs (human) for the earlier Hb nanocapsules(EncHb (10:1). This very significant increase was further improved usingstep-wise incremental formulations until we reached a maximalconcentration of 3.66 gm/dl(S.D.=0.03) and 24.2 hrs (rats) or 41.5 hrs(Human) to fall to the level of 1.67 gm/dl (eg. EncHb 1.5 Conc. (5K) toEncHb 1.5 Conc. (15K) XL).

(ii) Formulation “1.5 Conc (5k)”

A formulation “1.5 Conc (5k)” was prepared in a manner similar to thatof “EncapHb (17:1)” formulation above except that the concentration ofthe PLA-co-PEG was increased to 1.5 in order to have a thickernanocapsule membrane that would be slower to biodegrade, therebyallowing for a longer circulation time was prepared. As a result, afurther increase in circulation time was obtained. Two minutes afterinfusion, the maximal non-rbc Hb was 3.60 gm/dl(S.D.=0.01) compared to3.05 gm/dl (S.D.=0.04) for the earlier Hb nanocapsules (Table 3). Theslope of the disappearance was also much slower, but what is moreimportant is that it took 20.0 hrs (rats) 34.3 hrs (Human) for thenon-rbc Hb level to fall to the level of 1.67 gm/dl (FIG. 5).

(iii) Formulation “2.0 Conc (5k)”

A “2.0 Conc (5k)” formulation was prepared based on the above “1.5 Conc(5k)” formulation except that a higher concentration of polymer was usedto further improve the stability of the nanocapsule membrane. However,the Hb nanocapsules formed this way tended to aggregate and thereforewere not selected for testing in animal studies.

(iv) Formulation “1.0 Conc (5k)(XL-Ecap, XL4)”

Formulation “EncapHb (17:1)” was modified by adding glutaraldehyde tothe Hb nanocapsules suspension after they were formed, to provide a “1.0Conc (5k)(XL-Ecap, XL4)” Formulation. This formulation cross-linked anysurface Hb to further increase the membrane stability. The hemoglobinnanocapsules “encapHb (17:1)” as shown in Table 3 were further treatedas follows. The hemoglobin nanocapsules are exposed to a cross-linker,glutaraldehyde by adding this to the suspension. This results in thecross-linking of any Hb near the surface of the nanocapsules so as tofurther increase the nanocapsule membrane stability. The polymerizationof hemoglobin was stopped by adding 2M of lysine (at molar ratio oflysine/hemoglobin=100:1) after 24 hours. This approach also increasedthe circulation time to the same degree as when using 1.5 concentrationof the polymer in Formulation “1.5 Conc (5k)”. Thus, two minutes afterinfusion, the maximal non-rbc Hb was also significantly higher: 3.60gm/dl (S.D.=0.01) compared to 3.05 gm/dl (S.D.=0.04) for the earlier Hbnanocapsules (Table 3). The slope of the disappearance was also muchslower, but what is more important is that it took 20.3 hrs (rats) 34.8hrs (Human) for the non-rbc Hb level to fall to 1.67 gm/dl (Table 3;FIG. 5).

(v) Formulation “1.0 Conc (15k)”

We also looked at the use of a higher molecular weight PLA to increasethe stability of the Hb nanocapsules membrane. For this we replaced the5K PLA with a 15K PLA for the Formulation “EncapHb (17:1)”. As a result,a very significant increase in the circulation half-time of thepreparation was observed as compared to that prepared by Formulation“EncapHb (17:1)”. Thus, in two minutes after infusion, the maximalnon-rbc Hb was: 3.57 gm/dl (S.D.=0.05) compared to 3.05 gm/dl(S.D.=0.04) for the earlier Hb nanocapsules. The slope of thedisappearance was also much slower, but what is more important is thatit took 21.2 hrs (rat) and 36.3 hrs (human) for the non-rbc Hb level tofall to the level of 1.67 gm/dl (Table 3; FIG. 5).

(vi) Formulation “1.5 Conc (15k)”

We next looked at combining the use of a higher molecular weight PLA(15k) with a higher concentration of the polymer (1.5 times higher).This is done by using the same formulation as Formulation “1.0 Conc.(15k)” above except with a 1.5 concentration of the polymer. Thisformulation combines (1) using the specially prepared polyhemoglobinwith low percentage of single tetrameric hemoglobin; (2) using a 1.5×concentration of the PLA-co-PEG copolymer; and (3) using a highermolecular weight PLA (15k).

This resulted in a further significant increase in the circulation time.In two minutes after infusion, the maximal non-rbc Hb was: 3.6458gm/dl(S.D.=0.02) compared to 3.05 gm/dl (S.D.=0.04) for the earlier Hbnanocapsules. The slope of the disappearance was also much slower, butwhat is more important is that it took 23.3 hrs(rats) 39.9 hrs (Human)for the non-rbc Hb level to fall to the level of 1.67 gm/dl (Table 3;FIG. 5).

(vii) Formulation “1.5 Conc (15k)(XL-Ecap, XL4)”

Finally, we combined all the above ways of improving the circulationtime by: (1) using the specially prepared polyhemoglobin with lowpercentage of single tetrameric hemoglobin; (2) using a 1.5×concentration of the PLA-co-PEG copolymer; (3) using a higher molecularweight PLA (15K); and (4) further cross-linking of the Hb nanocapsuleswith glutaraldehyde.

The circulation time increased slightly from the above when only 3 ofthe above factors have been incorporated. Thus, in two minutes afterinfusion, the maximal non-rbc Hb was: 3.6583 gm/dl (S.D.=0.03) comparedto 3.05 gm/dl (S.D.=0.04) for the earlier Hb nanocapsules. The slope ofthe disappearance was also much slower, but what is more important isthat it took 24.2 hrs (rats) 41.5 hrs (Human) for the non-rbc Hb levelto fall to 1.67 gm/dl (Table 3; FIG. 5).

If we project this to human, a functional circulation time of the mostrecent Hb nanocapsules (Formulation “1.5 Conc (15k)(XL-Ecap, XL4)”) of41 hours can be expected. This is a very significant and importantincrease that provides longer function of encapsulated hemoglobin, andthus provides a promising alternative to donor blood. The Hbnanocapsules are likely to have even higher circulation time in humancompared to PolyHb. This is because the reticulo-endothelial system(RES) in rats is much more efficient in removing particulates likenanocapsules as compared to PolyHb solution. A further advantage ofpolyHb is that even if it leaks out after infusion it would continue toact and would not cause any adverse effects. It is fully contemplatedthat the nanocapsules of the present invention could serve as a usefulcarrier for other modified Hb including recombinant Hb, as well as othertherapeutically effective agents of interest.

EXAMPLE V Prevention and Conversion of Methemoglobin

With increased hemoglobin in circulation in the body at 37° C., there isa steady increase in the production of methemoglobin. Oxidation ofhemoglobin to methemoglobin inside red blood cell is prevented by theenzyme systems of the red blood cells. In the absence of these enzymes,methemoglobin is formed when the circulation time is increased.Accordingly, alternative embodiments of the present invention areprovided to prevent the accumulation of methemoglobin when hemoglobin isemployed with the nanocapsule compositions disclosed herein.

One embodiment of the present invention includes providing all of theenzymes normally present in the red blood cell together with hemoglobinin the nanocapsule. Another embodiment of the present invention providesa nanocapsule composition that is permeable to external factors such asascorbic acid, or glutathione which are naturally present in blood thatwould prevent the accumulation of methemoglobin in vivo. For example,the nanocapsule composition of the present invention can be prepared sothat it retains macromolecules like hemoglobin. At the same time, it canbe selectively permeable to smaller molecules like ascorbic acid orglutathione.

Currently, hemoglobin encapsulated in nanocapsules of the presentinvention, is changed slowly to methemoglobin after infusion into thebody at 37° C. Methemoglobin, unlike hemoglgobin, can no longer carryand deliver oxygen. If the nanocapsule membrane is made selectivelypermeable as described above, then while retaining hemoglobin, themembrane would allow ascorbic acid, glutathione or other similarsubstances from the circulating plasma to diffuse into the nanocapsules.Since these molecules help to prevent hemoglobin from changing tomethemoglobin, this enhances the ability of hemoglobin to carry anddelivery oxygen.

Hb nanocapsules were prepared to contain all the enzymes of the redblood cells. This was done by using the whole content of red blood cellsexcept the red blood cell membranes (hemolysate) extracted from redblood cells with all the enzymes and hemoglobin. For this we can use anyof the procedures described above for the formation of hemoglobinnanocapsules, especially those method that results in prolongedcirculation half-time. We prepared Hb nanocapsules with highermethemoglobin level of 7%. We then incubated these nanpcapsules at 37°C. and followed the changes in the % of metHb.

When suspended in Ringer lactate containing 100 mg/dl glucose, metHbincreased by 2.5% in 6 hours. Alternativley, when suspended in Ringerlactate containing 100 mg/dl glucose and 0.02 mM NADH, metHb increasedin the first hour as above. However, as glucose and NADH entered thenanocapsules to start the multi-enzyme reaction metHb decreased at arate of 1.5% in 5 hours. This result is very exiting because it showsthat we only need to encapsulate fresh red blood cell contents with thenormal amount of methemoglobin reductase system. This way, 100 mgglucose (available as blood glucose) and 0.02 mM NADH in the suspendingmedium not only prevent metHb formation, but also convert metHb back toHb. By further optimization of the NADH concentration, this can beincreased further.

When suspended in Ringer lactate with 100 mg/dl glucose and 0.02 mMNADPH, metHb increased at the same rate as when suspended in Ringerlactate containing 100 mg/dl glucose. This is because unlike NADH, thelarger cofactor NADPH is not permeable across the nanocapsules. SinceNADPH is not permeable, it can be enclosed inside the nanocapsules. Thisavoids the need to supply external cofactor and allows the reaction totake place like it does in the RBC. The circulating blood contains closeto 100 mg/dl of glucose that can enter the nanocapsules to act withNADPH and the multi-enzyme system. For RBC, blood glucose can enter theRBC by membrane transport.

There are substances in the plasma that prevents methemoglobinformation. Examples include ascorbic acid and glutathione. We suspendedHb nanocapsules of the present invention in solutions, of ascorbic acid,glutathione, or methylene blue. The Hb nanocapsule membrane waspermeable to all these factors. As a result, after infusion, the Hb inthe nanocapsules were exposed to factors in the circulating plasma thatprevent the formation of metHb. In accordance with one embodiment of thepresent invention, other materials usually present inside red bloodcells, including without limitation, enzymes such as catalase,superoxide dismutase and methemoglobin reductase, and cofactors amongstothers, may be encapsulated in addition to hemoglobin.

It is fully contemplated that the present invention is adaptable for thepreparation of nanocapsule compositions comprising therapeuticallyeffective concentrations of other agents of therapeutic interest,including macromolecules.

Accordingly, the nanocapsule compositions of the present invention mayinclude a variety of agents and/or molecules of therapeutic interest toeffect a desired result upon administration to a recipient. Furthermore,the nanocapsule compositions of the present invention may be selectivelypermeable to agents and/or molecules of therapeutic interest so as toallow the same to permeate the nanocapsule membrane and interact withthe encapsulated agent.

According to the present invention there is also provided an effectivedelivery system for delivering encapsulated agents into in vivocirculation. A delivery system adapted for the step-wise release of anagent of therapeutic interest in vivo is also provided. According tothis embodiment, a plurality of nanocapsule compositions each having apredetermined release rate for an encapsulated agent of therapeuticinterest may be administered simultaneously to achieve a controlledstep-wise release thereof in vivo.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth, and as follows in the scopeof the appended claims.

What is claimed is:
 1. A biodegradable polymeric nanocapsule compositionadaptable for use as an artificial red blood cell, said nanocapsuleencapsulating an agent of therapeutic interest; wherein said agent oftherapeutic interest is cross-linked to the interior surface of thenanocapsule and enhancing in vivo circulation time thereof, saidnanocapsule having a bilayer membrane defining an interior surface andan exterior surface and comprising a diblock copolymer consisting of apolylactic acid polymer block and a polyethylene glycol polymer block,wherein said nanocapsule membrane further comprises a lipid component,wherein said diblock copolymer is soluble in acetone and insoluble inwater and wherein said polyethylene glycol is present on both theinterior surface and the exterior surface of the membrane therebydefining a hydrophilic interior wherein said cross-linked agent oftherapeutic interest is present or is entrapped.
 2. The nanocapsulemembrane composition of claim 1 wherein said lipid component is providedin an amount less than each of said polylactic acid polymer andpolyethylene glycol.
 3. The nanocapsule membrane composition of claim 1wherein said polylactic acid polymer has a molecular weight of 5 KDa to25 KDa.
 4. The nanocapsule membrane composition of claim 3 wherein themolecular weight of said polylactic acid polymer is at least 10 KDa. 5.The nanocapsule membrane composition of claim 1 wherein saidpolyethylene glycol has a molecular weight of at least 2,000 Da.
 6. Thenanocapsule membrane composition of claim 5 wherein said polyethyleneglycol is methoxypolyethylene glycol.
 7. The nanocapsule membranecomposition of claim 1 having an in vivo circulation halftime of atleast 14 hours.
 8. The nanocapsule membrane composition of claim 1wherein said agent of therapeutic interest is a macromolecule.
 9. Thenanocapsule membrane composition of claim 8 wherein said macromoleculeis hemoglobin.
 10. The nanocapsule composition of claim 8 wherein saidagent of therapeutic interest is a cross-linked macromolecule chain. 11.The nanocapsule membrane composition of claim 1 further adapted toprovide a selectively permeable membrane for an encapsulated agent oftherapeutic interest.
 12. The nanocapsule membrane composition of claim1 wherein said lipid component is a phospholipid.
 13. The nanocapsulemembrane composition of claim 1 further adapted to maintain an agent oftherapeutic interest in vivo, with a circulation half-time of at least14 hours.
 14. A hemoglobin nanocapsule composition, said compositioncomprising a biodegradable polymeric nanocapsule membrane encapsulatinga therapeutically effective concentration of a hemoglobin preparation;wherein said hemoglobin preparation is cross-linked to the interiorsurface of the nanocapsule, said nanocapsule membrane comprising acopolymer of polylactic acid polymer and polyethylene glycol; saidcopolymer being soluble in acetone and insoluble in water; wherein saidnanocapsule membrane further comprises a lipid component; wherein saidnanocapsule composition is adaptable for enhancing in vivo circulationtime of said hemoglobin preparation.
 15. The hemoglobin nanocapsulecomposition of claim 14 wherein said lipid component is a phospholipid.16. The hemoglobin nanocapsule composition of claim 14 wherein saidlipid component is provided in an amount less than each of saidpolylactic acid polymer and polyethylene glycol.
 17. The hemoglobinnanocapsule composition of claim 14 wherein said polyethylene glycol ismethoxypolyethylene glycol.
 18. The hemoglobin nanocapsule compositionof claim 14 wherein said polylactic acid polymer has a molecular weightof 5 KDa to 25 KDa.
 19. The hemoglobin nanocapsule composition of claim18 wherein said hemoglobin is a polymerized hemoglobin.
 20. Thehemoglobin nanocapsule composition of claim 18, wherein theconcentration of the copolymer is increased to provide a nanocapsulemembrane having a greater thickness.
 21. The hemoglobin nanocapsulecomposition of claim 17 wherein said polyethylene glycol has a molecularweight of 2,000 Da.
 22. The hemoglobin nanocapsule composition of claim18 wherein the molecular weight of said polylactic acid polymer is15KDa.
 23. The hemoglobin nanocapsule composition of claim 14 furthercomprising at least one other agent known to inhibit the production ofmethemoglobin.
 24. The hemoglobin nanocapsule composition of claim 14further adapted to be selectively permeable to molecules present in invivo circulation; wherein said molecules inhibit the encapsulatedhemoglobin from being converted to methemoglobin.
 25. The hemoglobinnanocapsule composition of claim 14 wherein said nanocapsule compositionis adapted to provide said hemoglobin preparation with an in vivocirculation half-time of at least 14 hours.
 26. A method for preparing ananocapsule membrane composition comprising a polylactic acid andpolyethylene glycol diblock copolymer having an enhanced circulationtime in vivo, said method comprising: (a) heating a mixture of apolylactic acid polymer and polyethylene glycol (PLA-PEG); (b) adding anaqueous solution comprising an agent of therapeutic interest to saidmixture; (c) precipitating a copolymer from said aqueous solution toobtain a nanocapsule composition; (d) cross-linking said agent oftherapeutic interest to the interior of the nanocapsule; and (e)extracting the nanocapsule composition therefrom; wherein saidcomposition comprises a biodegradable, polymeric nanocapsule membraneencapsulating said agent of therapeutic interest.
 27. The method ofclaim 26 wherein said copolymer is soluble in acetone and insoluble inwater.
 28. The method of claim 26 wherein said nanocapsule membranefurther includes a lipid component.
 29. The method of claim 26 whereinsaid lipid component is a phospholipid.
 30. The method of claim 26wherein said step of heating is performed in the presence of a catalyst.31. The method of claim 30 wherein said catalyst isstannous-2-ethylhexanoate.
 32. A method according to claim 26 whereinthe PLA-PEG mixture includes a PLA component having a molecular weightof at least 10,000.
 33. The method of claim 32 wherein the PLA-PEGmixture further includes a PEG component having a molecular weight of atleast 2,000 Da.
 34. The method of claim 26 wherein said agent oftherapeutic interest is a macromolecule.
 35. The method of claim 34wherein said macromolecule is hemoglobin.
 36. The method of claim 34wherein said agent of interest is one of a protein, enzyme, gene, RNAfragment or DNA fragment.
 37. The method of claim 26 wherein saidpolylactic acid polymer has a molecular weight of 5 KDa to 25 KDa. 38.The method of claim 37 wherein said molecular weight is at least 10 KDa.39. The method of claim 26 wherein said step of heating said copolymermixture is carried out at at least 180 degrees Celcius.
 40. The methodof claim 38 wherein said molecular weight is 15 KDa to 25 KDa.
 41. Themethod of claim 40 wherein said step of heating said copolymer mixtureis carried out at 200 degrees Celcius.
 42. The method of claim 26wherein the circulation time of the encapsulated agent is also enhanced.43. A delivery system for enhancing the circulation time of an agent oftherapeutic interest in vivo, said system comprising a biodegradablepolymeric nanocapsule membrane composition comprising a diblockcopolymer membrane encapsulating said agent of therapeutic interest;wherein said agent of therapeutic interest is cross-linked to theinterior surface of the nanocapsule; wherein said copolymer membraneincludes a copolymer of polylactic acid and polyethylene glycol and issoluble in acetone and insoluble in water; wherein said nanocapsulemembrane comprises a lipid component: said copolymer membrane beingadaptable to deliver the encapsulated agent of therapeutic interest intoin vivo circulation at a controlled rate of release.
 44. The deliverysystem of claim 43 wherein said agent of therapeutic interest is amacromolecule.
 45. The delivery system of claim 44 wherein saidmacromolecule is hemoglobin.
 46. The delivery system of claim 43 whereinthe biodegradable polymeric nanocapsule composition is further adaptedto be selectively permeable to biological components present in in vivocirculation of a recipient.
 47. The delivery system of claim 43 whereinsaid nanocapsule composition is further adapted to encapsulate otherbiological components together with said agent of therapeutic interest.48. The delivery system of claim 43 wherein said lipid component is aphospholipid.
 49. A delivery system for providing step-wise release ofan agent of therapeutic interest in vivo, said system comprising aplurality of biodegradable polymeric nanocapsule compositions eachadapted to release an encapsulated agent of therapeutic interest at adifferent predetermined rate of release in vivo; wherein saidencapsulated agent of therapeutic interest is cross-linked to theinterior surface of said biodegradable polymeric nanocapsule; whereineach of said plurality of biodegradable polymeric nanocapsules includesa diblock copolymer membrane comprising a copolymer of polylactic acidand polyethylene glycol, said copolymer being soluble in acetone andinsoluble in water.
 50. The delivery system of claim 49 wherein saidagent of therapeutic interest is a macromolecule.
 51. The deliverysystem of claim 50 wherein said macromolecule is hemoglobin.
 52. Thedelivery system of claim 49 wherein said copolymer membrane furtherincludes a lipid component.
 53. The delivery system of claim 49 whereinthe biodegradable polymeric nanocapsule composition is further adaptedto be selectively permeable to biological components present in in vivocirculation of a recipient.
 54. The delivery system of claim 49 whereinsaid nanocapsule composition is further adapted to encapsulate otherbiological components together with said agent of therapeutic interest.55. The delivery system of claim 52 wherein said lipid component is aphospholipid.
 56. A nanocapsule composition comprising a biodegradablepolymeric nanocapsule membrane encapsulating a therapeutically effectiveconcentration of a macromolecule; wherein said macromolecule iscross-linked to the interior surface of the nanocapsule; saidnanocapsule membrane comprising a diblock copolymer of polylactic acidpolymer and polyethylene glycol; said copolymer being soluble in acetoneand insoluble in water; wherein said nanocapsule membrane comprises alipid component; wherein said nanocapsule composition is adaptable forenhancing the in vivo circulation time of said macromolecule.
 57. Thenanocapsule composition of claim 56 wherein said lipid component isprovided in an amount less than each of said polylactic acid polymer andpolyethylene glycol.
 58. The nanocapsule composition of claim 56 whereinsaid polyethylene glycol is methoxypolyethylene glycol.
 59. Thenanocapsule composition of claim 56 wherein said polylactic acid polymerhas a molecular weight of 5 KDa to 25 KDa.
 60. The nanocapsulecomposition of claim 59 wherein the a concentration of the copolymer isincreased to provide a nanocapsule membrane having a greater thickness.61. The nanocapsule composition of claim 58 wherein said polyethyleneglycol has a molecular weight of 2,000 Da
 62. The nanocapsulecomposition of claim 59 wherein the molecular weight of said polylacticacid polymer is 15 KDa.
 63. The nanocapsule composition of claim 56further adapted to be selectively permeable to molecules present in invivo circulation.
 64. The nanocapsule composition of claim 56 whereinsaid nanocapsule composition is adapted to maintain said macromoleculewith an in vivo circulation half-time of at least 14 hours.